Residues of organophosphate (OP) insecticides are undesirable contaminants of the environment and a range of commodities. Areas of particular sensitivity include contamination of soil, irrigation tailwater that is re-cycled, used by irrigators downstream or simply allowed to run off-farm, and residues above permissible levels in agricultural and horticultural exports. Poisoning with organophosphates presents a problem for agricultural workers that are exposed to these chemicals, as well as military personnel exposed to organophosphates used in chemical warfare. Furthermore, the stockpiling of organophosphorus nerve agents has resulted in the need to detoxify these stocks. Bioremediation strategies are therefore required for eliminating or reducing these organophosphate residues and/or stockpiles.
One proposed strategy involves the use of enzymes capable of immobilising or degrading the organophosphate residues. Such enzymes may be employed, for example, in bioreactors through which contaminated water could be passed, or in washing solutions after post-harvest disinfestation of fruit, vegetables or animal products to reduce residue levels and withholding times. Suitable enzymes for degrading organophosphate residues include OP hydrolases from bacteria (Mulbry, 1992; Mulbry and Kearney, 1991; Cheng et al., 1999; U.S. Pat. Nos. 5,484,728; 5,589,386; Dong et al., 2005), vertebrates (Wang et al., 1993; 1998; Gan et al, 1991; Broomfield et al., 1999) and OP resistant insects (WO 95/19440 and WO 97/19176). It is desirable that the OP hydrolases degrade the organophosphate residues at a rapid rate.
The most thoroughly studied OP degrading enzyme is bacterial organophosphate dihydrolase (termed OPD, OPH or PTE), which is encoded by identical genes on dissimilar plasmids in both Flavobacterium sp. ATCC 27551 and Brevundimonas diminuta MG (Harper et al., 1988; Mulbry and Karns, 1989). OPD is a homodimeric protein that is capable of hydrolysing a wide range of phosphate triesters (both oxon and thion OPs) (Dumas et al., 1989a, b). Its reaction mechanism directly or indirectly involves metal ions, preferably Co++, but also including Zn++, Cd++, Fe++ and other divalent cations. OPD has no detectable activity with phosphate monoesters or diesters (Dumas et al., 1989a, b; 1990).
OPD homologues (phosphotriesterase homology proteins, or PHPs) have been identified in the genomes of Escherichia coli (ePHP), Mycobacterium tuberculosis (mtPHP) and Mycoplasma pneumoniae (mpPHP), although only ePHP has been tested for phosphotriesterase activity (Scanlan and Reid, 1995; Buchbinder et al., 1998). No activity was detected in ePHP crude lysates with any of the substrates tested, such as p-nitrophenyl acetate, bis(p-nitrophenyl) phosphate, paraoxon and p-nitrophenyl phosphate. A class of more distantly related proteins has been identified that has very low levels of OP hydrolase activity, but high levels of lactonase activity, likewise the OPD enzymes have very low levels of lactonse activity (less than 0.001% of the activity against OP substrates) (Afriat et al., 2006).
OPD homologues have also been identified in vertebrates (Davies et al., 1997), although their function in these organisms is unknown. OPD, ePHP, mtPHP and mammalian PHPs are 27-30% identical at the amino acid level, while mpPHP is less similar. Amino acid residues involved in Zn++ binding are conserved across the all members of the phosphotriesterase family identified to date (Buchbinder et al., 1998).
More recently, an OP degrading enzyme has been isolated from Agrobacterium (WO 02/092803; Horne et al., 2002; Jackson et al., 2009). This enzyme was termed OpdA because it shares about 90% amino acid sequence identity with OPD. Despite the relatedness of OpdA and OPD at the amino acid level, these enzymes have been shown to have varying activities against different OPs.
There is a need for further OP degrading enzymes which can be used in bioremediation strategies. In particular, there is a need for enzymes with enhanced activity against specific OPs that can be used in bioremediation in the field.